p21-activated Kinases (PAKs) Mediate the Phosphorylation of PREX2 Protein to Initiate Feedback Inhibition of Rac1 GTPase

Abstract

Background:The role of phosphorylation for regulating the Rac1 GEF PREX2 is not understood. Results: PAK phosphorylation of PREX2 downstream of PIP 3 and G␤␥ reduces PREX2 GEF activity. Conclusion: Second messengers can initiate negative feedback to decrease Rac1 activation through PAK phosphorylation of PREX2. Significance: PAK negative regulation of GEFs could represent a broadly utilized mechanism to attenuate Rac1 activation and its outputs.

PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs)

“…1, F and G). AKT, which is activated by PIP 3 production, was significantly phosphorylated within the first 30 s. This time course of PREX1 phosphorylation is consistent with the time course of PAK-dependent PREX2 phosphorylation downstream of insulin receptor activation (33). Additionally, insulinmediated phosphorylation of PREX2 also requires PI3K, and because of these similarities between PREX2 and PREX1, we tested whether PREX1 phosphorylation downstream of RTKs is also mediated by PAKs.…”

“…Interestingly, our experiments show that the phosphorylation of PREX1 is not as sensitive to BEZ235 as it is to GDC0941, as the induced upper band is still faintly present after BEZ235 treatment, whereas the upper band is completely gone after treatment with GDC0941. Similarly, we previously reported that the phosphorylation of PREX2 downstream of insulin is more sensitive to GDC0941 than to BEZ235 (33). The reduced sensitivity of PREX phosphorylation to BEZ235 could be because BEZ235 is a less potent inhibitor of PI3K␤, and PI3K␤ can help to maintain higher PIP 3 levels when PI3K␣ is efficiently inhibited.…”

“…In Vitro PAK2 Kinase Assay-His 6 -PAK2 was purified as described previously (33). For the kinase assay, MYC PREX1 or V5 PREX2-expressing HEK293 cells were starved and then treated with 500 nM GDC0941 and 5 M PF-3758309 for 30 min.…”

“…We have shown previously that insulin-mediated phosphorylation of PREX2 reduces binding to PIP 3 to prevent PIP 3 -dependent activation of GEF activity (33). Furthermore, multiple studies on PREX1 have shown that phosphorylation can affect GEF activity.…”

“…PAK-dependent phosphorylation of PREX2, which occurs downstream of Rac activation, reduces PIP 3 and G␤␥ binding as well as PIP 3 and G␤␥-stimulated PREX2 GEF activity, suggesting that these phosphorylation events are part of a negative feedback circuit to reduce Rac activation after insulin stimulation (33). In this study, we found that PREX1 is phosphorylated after insulin treatment of breast cancer cells and that insulin, neuregulin, and IGF1-mediated phosphorylation of PREX1 are PI3K-and PAK-dependent.…”